Prism design for scanning applications and illumination of microscopy sample

ABSTRACT

There is disclosed a prism for use in scanning applications such as total internal reflection microscopy in which the prism is translated relative to an incident light beam. A geometry is disclosed which cancels walk of the beam footprint at the base of the prism. Walk of the beam footprint due to irregularities in a largely planar sample surface located at the prism base are cancelled by coupling movement of the incident light beam to movement of the sample in the field of view of an objective lens, for example as part of an autofocus arrangement.

REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of International ApplicationPCT/GB2003/000154 with International filing date of Jan. 15, 2003.

FIELD OF THE INVENTION

The present invention relates to scanning applications in which asample, together with an angular optic such as a triangular ortrapezoidal prism, are scanned in a fixed laboratory frame of reference.The fixed laboratory frame is defined by a fixed light source generatinga fixed light beam and a detector. The angular optic couples the lightbeam to the base interface of the angular optic where the sample islocated, such that the beam is incident on the base interface at an offnormal angle. In such applications it is desirable that the intercept ofthe light beam at the base interface of the angular optic (the“footprint”) remains stationary in the laboratory frame as the optic andsample are scanned so that there is no loss of image integrity at thedetector.

The present invention also relates to scanning applications involvingimaging systems and techniques in which an illumination beam of light isdirected to a largely planar sample at an oblique angle.

BACKGROUND OF THE INVENTION

One example application is Total Internal Reflection Microscopy, whichis a technique for observing samples illuminated by an evanescent wave.Total internal reflection occurs when a beam of light travelling througha very dense medium such as glass encounters an interface with a lessdense medium such as air or water, at an angle to the normal which isgreater than the critical angle for the interface. The critical anglefor a glass/water interface is given by Fresnel's Law of Refraction as:

θ_(c)=sin⁻¹ (n _(water) /n _(glass))

At angles greater than the critical angle, when total internalreflection takes place, an electric field component of the lightpenetrates through the interface into the water as an evanescent wave.The evanescent wave has the same wavelength as the incident beam butpenetrates only a very short distance into the water, typically no morethan 1 μm. The evanescent wave decays exponentially from the interfaceinto the water with a characteristic penetration depth dependent on thewavelength and angle of incidence of the totally internally reflectedlight.

In Total Internal Reflection Fluorescence Microscopy, fluorophores maybe excited by the light in the evanescent field if they are close to theglass/water interface, but fluorophores further away in the bulk of thesolution will not be excited. The result is that images with very lowbackground fluorescence are obtained. FIG. 1 shows a typical instrumentset up used in Total Internal Reflection Fluorescence Microscopy. Asample is placed such that it is located directly on the interface ofthe base of a light coupling optic or dispersion prism 1. Alternatively,a glass slide 2 may be optically matched to the prism, and the samplelocated on the base of the slide. Total internal reflection then occursat the base of the slide. Typically, the objective lens 3 and externallight source 4 are fixed in the lab frame and the sample on which thelight coupling optic or prism is fixed is scanned in a planeperpendicular to the objective lens axis. The prism 1 therefore movesrelative to the objective lens 3 and the light source 4. Conventionallya 45° or 60° dispersion prism is used, but to obtain light beamsincident on the base of the prism at angles close to and greater thanthe critical angle, the light must usually be incident on the input faceof the prism at off normal angles of incidence to achieve refraction ofthe beam at the air/glass interface. The deviation of the beam causesthe reflection footprint at the base of the prism 1 to walk db as theprism is translated dx towards or away from the light source. In alimiting case light propagating parallel to the prism base will berefracted such that the footprint at the prism base moves equally and inthe same direction as the prism (db/dx=0). In this case the footprintmoves dx in the lab frame and the illuminated area moves rapidly awayfrom the imaging lens as the sample is scanned.

In imaging systems in which excitation or illumination of a sample islargely confined to a sample plane, such as in total internal reflectionfluorescence microscopy (TIRFM), accurate excitation and imaging ofmaterial in this plane can be extremely sensitive to movements orirregularities of the plane. This is especially true in systems of highmagnification, since a higher magnification generally results in asmaller field of view and depth of focus. If the sample is scanned,replaced or otherwise moved, adjustments need to be made to keep theimage in focus. If the illumination beam is obliquely incident on thesample plane, as it is in TIRFM, irregularities or movements in thesample plane cause the intersection, or footprint of the beam in theplane to move laterally across the imaging area. This effect may betermed footprint misalignment, and results in the objective or imaginglens looking at a different part of the sample plane to that which isbeing illuminated.

Variations and irregularities may be present across a particular sample,causing footprint misalignment as the sample is scanned. Variations mayalso arise between consecutive samples, making an initial alignment ofthe illumination beam necessary when a new sample is loaded or set up.

Footprint misalignment has been found to be a particular problem insetting up a new sample for scanning with the TIRFM technique. In FIG.2, the TIRFM arrangement of FIG. 1 is represented in section. Anillumination laser beam 5 is transmitted through the prism 1, beforepassing through a layer of index matching fluid into the sample slide 2.The thickness of the slide in the drawing has been greatly exaggeratedfor clarity. The sample plane is defined by the lower surface of theslide. The angle of incidence of the beam onto the lower surface of theslide is sufficiently oblique that total internal refraction takesplace, and none of the illumination beam propagates through the bottomof the slide 2. For a glass-water interface an angle of incidence ofabout 68° is generally appropriate for ensuring total internalreflection of all components of the beam.

In the unaligned arrangement shown in FIG. 2, the focal plane 10 of theobjective lens 3 lies within the slide 2, and the illumination footprint12 lies to the left of the field of view of the objective lens 3.Approximate focus of the objective on the sample 14 can be achieved by,for example, observing the geometry of a drop of immersion oil betweenthe objective lens 3 and a cover slip placed over the sample 14, thusbringing the focal plane 10 roughly into coincidence with the sample 14.Approximate alignment of the footprint 12 with a region of interest ofthe sample surface being imaged using the objective lens can then beachieved by adjusting the illumination laser beam 5 and observing thescattered light until a high contrast background is observed, as long asthe focus is not too far from correct. This high contrast backgroundoriginates from point defects and irregularities in the surface and maybe described as a grainy image, typically including bright circularrings which may consist of intermittent bright and dark rings (“airydiscs”) when slightly defocussed or a bright point when in focus.Finding and correctly identifying this grainy image is difficult. If thefootprint 12 is too far from the objective lens field of view, which maybe very small, a largely blank, or at least less grainy image willresult. If a bubble is present in either the immersion oil or indexmatching fluid or if light reflects from the rim of the objective lensaperture 3 then the image may become swamped with scattered light.

When a grainy image has been found, the objective lens 3 is translatedtowards or away from the sample 14 until a scratch or point defect onthe slide-sample interface comes into focus, at the same time adjustingthe footprint to complete the alignment. This process is often hamperedby the presence of strong scatter, and is made more difficult becausethe focus in a first dimension and the footprint position in the twoother dimensions need to be adjusted at the same time. The processbecomes particularly difficult at high magnifications andcorrespondingly small depths of focus and small fields of view. Theseset-up difficulties present significant obstacles when designing TIRFMor similar systems which are suitable for automated or semi-automatedhigh throughput and/or scanning applications. When the sample isscanned, irregularities in the sample surface, even if compensated forusing an autofocus arrangement, can still result in footprintmisalignment.

OBJECTS OF THE INVENTION

It is an object of the present invention to obtain a footprint which isstatic in the laboratory frame of reference, defined by the objectivelens and light source, such that the area illuminated at theglass/aqueous interface does not move away from the optical axis of thelens as the sample and prism are scanned.

It is also an object of the invention to address the difficultiesencountered in aligning and focussing the optics in imaging systems inwhich a largely planar sample is illuminated at an oblique angle.

It is also an object of the invention to reduce misalignments andfocussing problems when an obliquely illuminated sample is scanned.

SUMMARY OF THE INVENTION

The invention addresses the problems of the related prior art byproviding a prism having particular advantageous properties, andapparatus and methods using such a prism. The invention also addressesproblems of the related prior art by providing correction of anillumination footprint by adjusting an optical path synchronously withchanges in the position of a sample surface or with the position of anelement which moves synchronously with the sample surface, with whichthe prism of the invention may advantageously be combined.

In particular, the invention provides a scanning apparatus whichcomprises:

a light source for generating a light beam; and

an angular prism coupled to a sample at a base interface;

characterised in that the base angle θ_(p) of the prism satisfies theequations:

$\begin{matrix}{{\frac{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )( {{\tan \; \theta_{i}\sin \; \theta_{p}} + {\cos \; \theta_{p}}} )}{\cos \; \theta_{c}{\cos ( {\theta_{p} \pm \theta_{c}} )}} = 1}{and}} & ( {{Eqn}\mspace{14mu} 1} ) \\{{n_{i}\sin \; \theta_{i}} = {n_{p}{\sin ( {\theta_{p} - \theta_{c}} )}}} & ( {{Eqn}\mspace{14mu} 2} )\end{matrix}$

wherein θ_(c) is the coupling angle required for light incident at thebase interface of the prism, θ_(i) is the incident angle of the lightbeam on the prism, n_(i) is the refractive index of the medium at theinterface where the light beam enters the prism and n_(p) is therefractive index of the prism.

A scanning method according to the invention comprises the steps of:

generating a light beam;

providing an angular prism in the path of the light beam, the prismbeing coupled to a sample at a base interface; and

moving the prism and sample relative to the light beam;

characterised in that the base angle θ_(p) of the prism satisfies theequations:

$\begin{matrix}{{\frac{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )( {{\tan \; \theta_{i}\sin \; \theta_{p}} + {\cos \; \theta_{p}}} )}{\cos \; \theta_{c}{\cos ( {\theta_{p} \pm \theta_{c}} )}} = 1}{and}} & ( {{Eqn}\mspace{14mu} 1} ) \\{{n_{i}\sin \; \theta_{i}} = {n_{p}{\sin ( {\theta_{p} - \theta_{c}} )}}} & ( {{Eqn}\mspace{14mu} 2} )\end{matrix}$

wherein θ_(c) is the coupling angle required for light incident at thebase interface of the prism, θ_(i) is the incident angle of the lightbeam on the prism, n_(i) is the refractive index of the medium at theinterface where the light beam enters the prism and n_(p) is therefractive index of the prism.

It has been found that, if the prism satisfies the above criteria, asolution exists where the footprint of the light beam on the base of theprism walks in an equal and opposite direction to the prism'stranslation in the lab frame i.e. db=−dx. Therefore, the point where thelight incident to the prism intercepts the prism base is fixed in thelab frame. This results in scanning of the sample without movement ofthe footprint with respect to a detector fixed in the lab frame andhence no loss of image integrity.

A solution is found wherein θ_(p)=θ_(c) and θ_(i)=0.

Preferably, the apparatus comprises a total internal reflectionmicroscopy apparatus and includes means for detecting interaction of thesample at the base of the prism or optically matched slide with anevanescent wave formed by total internal reflection of the light beam atthe base of the prism or at the base of a slide which is opticallymatched to the prism. Preferably, the method according to the presentinvention includes the step of detecting interaction of a sample at thebase of the prism with an evanescent wave formed by total internalreflection of the light beam at the base of the prism.

For light entering the prism from air, n_(i)=1, and therefore

sin θ_(t) =n _(p) sin (θ_(p)−θ_(c))

For total internal reflection to occur at the base interface θ_(c) mustbe greater than or equal to the critical angle for the interface ie

θ_(c)≧sin⁻¹ (n _(s) /n _(p))

Wherein n_(s) is the refractive index of the sample medium.

In total internal reflection microscopy, it is preferable that thecoupling angle is greater than but close to the critical angle as thismaximises the penetration of the evanescent wave into the sample medium.It is generally preferable that θ_(c) is slightly above the criticalangle because, although penetration of the evanescent wave is at amaximum at the critical angle, there will be a spread of angles withinthe beam and, to ensure total internal reflection of the entire beam itis preferable to have θ_(c) slightly above the critical angle.

This also accommodates minor variations in the refractive indices of theinterfacial media.

For a quartz/water interface at the base of the prism, where n_(p)=1.46,the critical angle of the base interface is 66°. A preferred value forθ_(c) would be 68°. A unique solution is found wherein θ_(i)=0, andθ_(p)=68°.

The invention also provides apparatus for imaging a sample, comprising:

an objective lens having a focal plane and an optical axis, the lensbeing arranged to collect light from a region of interest definedrelative to said optical axis;

an illumination director controllable to direct a beam of light along anoptical path so as to illuminate the sample in the region of interest atan angle oblique to said optical axis; and

an illumination director controller arranged to control the illuminationdirector relative to the sample such that the optical path remainsdirected to the sample in the region of interest following replacementof the sample, or movement of the sample relative to the objective lens.

The beam of light may be directed onto the sample through a prism havinga base angle as set out above, in particular when the invention isapplied in the construction or use of a total internal reflectionmicroscope.

The term “objective lens” is to be understood as covering suitablecompound lens arrangements and ancillary parts of any suitable objectivelens assembly including non-optical parts for mounting, focussing andotherwise controlling the lens.

The illumination beam may be directed at the sample continuously. Morepreferably, the beam is shuttered or otherwise turned off or blocked forsome or most of the time, for example to avoid photo bleaching of thesample.

The illumination director may, for example, be a mirror suitably mountedto cause the footprint of the beam of light on the sample to track theregion of interest, thereby reducing or eliminating the need to makemanual adjustments to the illumination director as the sample isscanned, or otherwise moved. The region of interest may cover the wholeor only a part of the field of view of the objective lens, because theapparatus may be used to image, at any instant, the whole or only a partof the sample within the field of view. Usually, the region of interestwill be a central portion of the field of view, relative to the opticalaxis of the objective lens, and could be represented as a single pointin the field of view fixed, or if required moveable, relative to theoptical axis.

The illumination director controller is preferably arranged toautomatically adjust the illumination director and/or the optical pathsynchronously with movements parallel to the optical axis of a part ofthe sample which is within the region of interest. This could beachieved, for example, by tracking the sample surface with a rangefinding device and using the output of the range finding device to driveadjustment of the illumination director.

In preferred embodiments, the illumination director controller isarranged such that the position, and preferably also the orientation ofthe illumination director relative to the part of the sample which iscurrently in the region of interest remains substantially constant.

The illumination director controller may be arranged to adjust theillumination director and/or optical path synchronously with movementsof the focal plane relative to the sample such that the beam of lightremains directed to the intersection of said region of interest and thefocal plane of the objective lens.

Preferred embodiments comprise an autofocus arranged such that the focalplane of the objective lens tracks the part of the sample which is inthe region of interest. In this way, the focal plane of the objectivelens and the viewed or relevant part of the sample remain coincidentsuch that adjusting the illumination director with respect to one givesrise to adjustment with respect to the other. Typically, the autofocuswill include a z-axis drive for adjusting the position of either thesample or objective lens along the optical axis of the lens. Such drivesare well known in the art.

Preferably, at least a part of the objective lens is moveable in adirection parallel to its optical axis so as to bring the part of thesample which is within the field of view or region of interest intofocus. The illumination director controller may then comprise a couplingarranged to adjust the illumination director synchronously with themovement of the at least part of the objective along the optical axis.

In particular, the coupling may be a direct mechanical coupling, such asa rigid bracket or other component, or combination of linked components.If an autofocus system is employed which uses a piezoelectric transducerto focus the objective lens, or similar low power arrangement, then itmay be desirable to use a lightweight bracket to avoid overloading thefocussing system. Alternatively, an electrical coupling could be used,for example by driving the objective lens and illumination director withsimilar actuators and a common signal.

Preferably, the objective lens is positioned on a first detector side ofthe sample, and the illumination director is positioned on the opposingillumination side of the sample, commonly referred to as transmissionmode. However, the invention is also applicable to so-called reflectionmode arrangements in which the beam approaches the sample from the sameside as the objective.

In preferred embodiments the apparatus is a total internal reflectionfluorescence microscope, in which the illumination beam reflects from aninterface adjacent to the sample.

Preferably, the illumination director is or comprises a mirror.Preferably also, the optical path of the illumination beam is incidenton this mirror in a direction parallel to the optical axis of theobjective lens. A fine adjust mechanism, for preliminary, manual or adhoc adjustments, may be provided on the mounting of this mirror.Preferably, however, to avoid undue vibration and consequent motion ofthe beam, the fine adjust mechanism is provided on a second mirrorfurther upstream from the sample along the beam, which may more easilybe rigidly mounted. The second mirror then directs the illumination beamonto the first mirror. Other arrangements of mirrors could be used.

Alternatively, the illumination director may be provided by thetermination of a light guide such as an optical fibre or fibre bundle,with optics incorporated as appropriate.

The invention also provides a method of automatically correcting theillumination footprint of the optical path of a light beam on thesurface of a microscopy sample, comprising the step of adjusting theposition of the optical path at the sample synchronously with changes inthe position of the sample surface or the position of an element whichmoves synchronously with the sample surface.

Conveniently, the element which moves synchronously with the samplesurface may be at least a part of an objective lens assembly arranged toform an image of the sample surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows part of an apparatus for total internal reflectionmicroscopy;

FIG. 2 illustrates the problem of achieving simultaneous objective focusand illumination beam footprint alignment in a total internal reflectionfluorescence microscope;

FIG. 3 is a schematic showing the total internal reflection footprintwalking with the displacement of the prism;

FIG. 4 is a graph of db/dx against prism angle for a quartz/water baseinterface and a coupling angle of 68°;

FIG. 5 shows an apparatus embodying the invention;

FIG. 6 shows a total internal reflection fluorescence microscope (TIRFM)embodying the invention;

FIG. 7 shows a range finding arrangement for use with the microscope ofFIG. 5 to provide an autofocus; and

FIGS. 8 a, 8 b and 8 c illustrate the footprint alignment methodeffected by the arrangement of FIG. 6.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 illustrates an apparatus for Total Internal ReflectionFluorescence Microscopy having a light source 4 which generates a beam 5which is incident on a prism 1 and is totally internally reflected atthe base interface of the prism. A sample is positioned adjacent to abase interface of the prism 1 and the evanescent wave interacts with thesample, producing fluorescence. The fluorescence passes through anobjective lens 3 and is directed towards a CCD camera 6 by a mirror 7,passing through a filter 8.

As shown in FIG. 3, the light beam 5 is incident on the prism 1 at θ_(i)to the normal, is refracted as it enters the prism at θ_(r) to thenormal and is incident on the base of the prism 1 at a coupling angleθ_(c), forming a footprint at the base interface. For total internalreflection to occur, θ_(c) must be at least the critical angle for thebase interface. As the prism moves dx in the lab frame to position 1¹,the footprint walks db in the prism frame.

The magnitude of the differential dl/db may be derived as follows:

${{Sine}\mspace{14mu} {rule}\mspace{14mu} \frac{l}{\sin ( {90 - \theta_{c}} )}} = \frac{r}{\sin \; \theta_{p}}$or r cos  θ_(c) = l sin  θ pCosine  rule  r² = b² + l² − 2 bl cos  θ_(p)(1)  in  (2)  l²sin²θ_(p) = cos²θ_(c)(b² + l² − 2 bl cos  θ_(p))quadratic  in  l  l²(cos²θ_(c) − sin²θ_(p)) − 2 bl cos  θ_(p)cos²θ_(c) + b²cos²θ_(c) = 0

solution to which is

$l = \frac{{2\; b\; \cos \; \theta_{p}\cos^{2}\theta_{c}} + \sqrt{\begin{matrix}{{4\; b^{2}\cos^{4}\theta_{c}\sin^{2}\theta_{p}} -} \\{4\; b^{2}\cos^{2}{\theta_{c}( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}}\end{matrix}}}{2( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

expand contents of the square root

$l = \frac{{2\; b\; \cos \; \theta_{p}\cos^{2}\theta_{c}} + \sqrt{\begin{matrix}{{4\; b^{2}\cos^{4}\theta_{c}\sin^{2}\theta_{p}} -} \\{{4\; b^{2}\cos^{4}\theta_{c}} + {4\; b^{2}\cos^{2}\theta_{c}\sin^{2}\theta_{p}}}\end{matrix}}}{2( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

take out 4b² cos²θ_(c) common in the square root

$l = \frac{{2\; b\; \cos \; \theta_{p}\cos^{2}\theta_{c}} + {2\; b\; \cos \; \theta_{c}\sqrt{\begin{matrix}{{\cos^{2}\theta_{c}\sin^{2}\theta_{p}} -} \\{{\cos^{2}\theta_{c}} + {\sin^{2}\theta_{p}}}\end{matrix}}}}{2( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

factorizing gives

$l = \frac{b\; \cos \; {\theta_{c}( {{\cos \; \theta_{p}\; \cos \; \theta_{c}} + \sqrt{ {{\cos^{2}\theta_{c}\cos^{2}\theta_{p}} - {\cos^{2}\theta_{c}} + {\sin^{2}\theta_{p}}} )}} }}{ {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

differentiating w.r.t. b

$\frac{l}{b} = \frac{\cos \; {\theta_{c}( {{\cos \; \theta_{p}\; \cos \; \theta_{c}} \pm \sqrt{ {{\cos^{2}\theta_{c}\cos^{2}\theta_{p}} - {\cos^{2}\theta_{c}} + {\sin^{2}\theta_{p^{r}}}} )}} }}{ {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

further factorization gives

$\frac{l}{b} = \frac{\cos \; {\theta_{c}( {{\cos \; \theta_{p}\; \cos \; \theta_{c}} \pm \sqrt{ {{\cos^{2}{\theta_{c}( {{\cos^{2}\theta_{p}} - 1} )}} + {\sin^{2}\theta_{p}}} )}} }}{ {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{\cdot p}}} )}$

recognising cos²θ_(p)−1=−sin²θ_(p) and factorising

$\frac{l}{b} = \frac{\cos \; {\theta_{c}( {{\cos \; \theta_{p}\; \cos \; \theta_{c}} \pm \sqrt{\sin^{2}{\theta_{p}( {1 - {\cos^{2}\theta_{c}}} )}}} }}{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

recognising 1−cos²θ_(c)=sin²θ_(c) and rooting the square

$\frac{l}{b} = \frac{\cos \; {\theta_{c}( {{\cos \; \theta_{p}\; \cos \; \theta_{c}} \pm {\sin \; \theta_{p}\sin \; \theta_{c}}} )}}{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

using trigonometric identity the differential simplifies to

$\frac{l}{b} = \frac{\cos \; \theta_{c}{\cos( \; {\theta_{p} \pm \; \theta_{c}} )}}{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}$

The magnitude of the differential dl/dx may be derived as follows:

${{Sine}\mspace{14mu} {rule}\mspace{14mu} \frac{l}{\sin ( {90 - \theta_{p} + \theta_{i}} )}} = \frac{x}{\sin \; ( {180 - \theta_{p} - ( {90 - \theta_{p} + \theta_{i}} )} }$Simplifies  to  l sin (90 − θ_(i)) = x  sin (90 − θ_(p) + θ_(i))l cos  θ_(i) = x cos (θ_(p) − θ_(i))$\frac{l}{x} = \frac{{\cos \; \theta_{p}\cos \; \theta_{i}} + {\sin \; \theta_{p}\sin \; \theta_{i}}}{\cos \; \theta_{i}}$$\frac{l}{x} = {{\cos \; \theta_{p}} + {\tan \; \theta_{i}\sin \; \theta_{p}}}$

Recognising that a translation of the prism dx in the positive xdirection results in a displacement db of the footprint in the negativedirection and by using the chain rule

$\frac{- {b}}{x} = {\frac{l}{x} \times \frac{b}{l}}$ so$\frac{b}{x} = \frac{{- ( {{\cos \; \theta_{p}} + {\tan \; \theta_{i}\sin \; \theta_{p}}} )} \times ( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )}{\cos \; \theta_{c}{\cos ( {\theta_{p} \pm \theta_{c}} )}}$

where θ_(i)=sin⁻¹ (n_(p) sin(θ_(p)−θ_(c)) from Fresnel's equation, forlight entering the prism from air, (n_(i.)=1).

For the footprint to remain stationary in the lab frame, it must walk inan equal and opposite direction in the prism frame to the prism'stranslation in the lab frame, i.e. db/dx=−1.

In total internal reflection microscopy, it is preferred that thecoupling angle is greater than but close to the critical angle as thismaximises the penetration of the evanescent wave into the sample medium,and it is generally preferable that θ_(c) is slightly above the criticalangle because, although penetration of the evanescent wave is at amaximum at the critical angle, there will be a spread of angles withinthe beam and, to ensure total internal reflection of the entire beam, itis preferable to have θ_(c) slightly above the critical angle.

For a quartz/water interface at the base of the prism, where n_(p)=1.46,the critical angle of the base interface is 66°. A preferred value forθ_(c) would be 68°. FIG. 4 shows the degree of walking of the footprintwith prism displacement as a function of the internal angle θ_(p) of theprism for θ_(c)=68°. The prism angle required for db/dx=−1 is 68° andthe light will be incident normal to the input surface of the prism,i.e. θ_(i)=0.

FIG. 5 shows a further apparatus embodying the invention which could be,for example, a total internal reflection microscope as set out above. Anobjective lens 40 has an optical axis 42 and a focal plane 44. A regionof interest 46 of the field of view of the objective lens 40 is defined.Usually the region of interest 46 will be a fixed central region of thefield of view, and is representative of the region of the field of viewwhich is to be studied, imaged or analysed. A sample (not shown) isplaced in or close to the focal plane 44 the lens and may be scannedrelative to the lens 40 to enable a wider area of the sample to bestudied.

A z-axis drive 48 is provided to move the lens 40 and/or sample alongthe optical axis 42 in order to keep the sample in focus. The z-axisdrive may be controlled using autofocus logic 50, which may, forexample, be responsive to a range finding mechanism output.

An illumination director 52 directs a beam of light 54 along acorresponding optical path towards the region of interest 46 within thefocal plane 44, so as to illuminate the sample in the region of interest46 at an angle oblique to the optical axis 42. Because of the obliqueincident angle of the beam, relative displacement of the sample and thebeam of light 54 along the optical axis 42 results in a lateraltranslation of the footprint of the beam of light 54 at the sample.Therefore, an illumination director controller 56 is provided to controlthe illumination director such that the beam of light continues toilluminate the sample in the region of interest when sample is replacedor moved relative to the objective lens 40 and/or optical path of thebeam of light 54.

Referring to FIG. 6 there is shown, schematically, a more specificarrangement 100, incorporating some of the features of FIGS. 1 and 2,for carrying out total internal reflection fluorescence microscopy,although for convenience and clarity, different reference numerals areused. A laser 102 and associated optics, mounted on a rigid opticaltable 104, form ar. illumination beam 106 which is directed along anoptical path by reflection from a source mirror 108 and then a targetmirror 110 to enter a side face of a prism 112 at an angle of incidencewhich is preferably approximately normal to the prism face. The sourcemirror is mounted on the table 104 and is provided with a manualadjustment mechanism. The target mirror is one possible embodiment ofwhat may more generally be termed an “illumination director”.

One face of a microscope slide 114 makes good optical contact with thebottom face of the prism 112, for example by means of a layer of indexmatching fluid. A sample 116 to be imaged forms a layer on the otherface of the microscope slide. The sample will typically be immersed inan aqueous solution. A transparent cover slip 117 overlies and protectsthe sample 16.

The critical angle for total internal reflection at the boundary betweenthe microscope slide 114 and the aqueous solution in which the sample116 is immersed is typically about 64° from a normal to the surface ofthe slide, the precise angle depending on the values of the refractiveindices of the slide and the solution. To ensure all components of theillumination beam 106 undergo total internal reflection at thisboundary, and to allow for the optical effects of various solvents andtemperature changes of the solvents, the target mirror 110 is arrangedsuch that the beam is incident on this boundary at a sample angle ofincidence of about 680 from the normal.

In addition to the mechanism of footprint misalignment caused byirregularities in the sample surface, illumination footprint walking canalso arise if the illumination beam propagates along a longer or shorteroptical path through the prism as the prism and sample are scanned. Tominimise this contribution to footprint walking, the prism is preferablyaligned such that the illumination beam enters the prism normal to theface of entry, and a prism having a base angle matching the desiredsample angle of incidence is preferably used, as set out above. Inparticular, the apparatus is preferably arranged such that the prism andlight beam satisfy the conditions set out in equations 1 and 2 above.

A triangular prism need not be used in embodiments of the invention,although preferred optics have the relationship between the face atwhich the illumination beam enters the optic and the face at which thedesired total internal reflection takes place discussed above. Clearly,a variety of different optic geometries can satisfy the functionalrequirements, such as truncated triangular prisms and so on, and theterm “prism” as used in this document should be understood to includeall such optic geometries.

Although the illumination beam undergoes total internal refraction atthe slide-sample boundary 114-116, an evanescent component of the beamis able to stimulate fluorescence in sample material lying within aboutone micrometer of the boundary. This fluorescence is collected using anobjective lens 118 which forms an image of the illuminated part of thesample 116 using a CCD camera or other low light level detector. Theimage is displayed on monitor 120.

One or more optical filters may be located between the sample and theobjective lens in order to eliminate any light from the illuminationbeam which may have scattered through the sample, for example off pointdefects in the prism or sample slide. Preferably, the filter or filterspass the maximum amount of light at the frequency of fluorescence of thesample while passing a minimum of light at the excitation frequency.

The region of the field of view of the objective lens 18 which is to beused for imaging the sample 116 may be referred to as the “region ofinterest”. It is, of course, desirable that this region should remainwithin or coincident with the footprint of the illumination beam despiterelative movements of the sample with respect to the objective lensand/or the target mirror 110. This region of interest may be a small orlarge part of the field of view of the objective lens, and may becentral or offset, but will preferably be fixed relative to the opticalaxis of the objective lens.

Focus of the image of the sample 116, in particular within the abovementioned region of interest, is achieved by translation of theobjective lens 118, or a part of the objective lens towards or away fromthe sample along its optical axis, which is generally perpendicular tothe focal plane of the objective, using a z-axis drive 119. A bracket122, which is preferably lightweight to avoid straining the z-axis drive119, which may incorporate a motor or other transducer, rigidly couplesthe objective lens 118 to the target mirror 110, so that as theobjective lens moves parallel to its optical axis the relative positionsof the target mirror 110 and objective lens 118 are fixed. Additionally,the source mirror 108 is arranged such that the illumination beam 106 isincident on the target mirror 110 in a direction substantially parallelto the focussing axis or perpendicular to the objective lens focal planeand sample-slide interface. The bracket 122 is one possible embodimentof what may more generally be termed an “illumination directorcontroller”.

The sample 116 is kept in focus by means of an auto focus mechanismwhich causes the objective lens to move as described above, for examplean auto focus mechanism as set out in copending U.K. patent application0200844.9. The prism 112, slide 114 and sample 116 are mounted on an x-ystage (not shown) so that the sample can be scanned across the field ofview of the objective lens 118, although the sample could of course befixed and the objective moved instead along with the target mirror andother parts of the illumination optics as required.

As the sample is scanned, the objective lens generally needs to becontinually refocused to adapt for irregularities, such as minorundulations, in the slide surface which cause the part of the samplewithin the field of view, and in particular the above mentioned regionof interest, to move towards or away from the objective lens. Thelightweight bracket 122 couples the focussing motions of the z-axisdrive 119, under the control of the autofocus mechanism, to the targetmirror 110, so that the footprint of the illumination beam 106 on thesample tracks the region of interest defined relative to the objectivelens 116.

The auto focus mechanism may use a range-finding light beam directedthrough the objective lens 118, but offset or displaced from the centraloptical axis. The range-finding beam is incident on and reflects fromthe sample surface in or close to the region of interest, although thebeam partially reflected from a surface parallel to the sample, such ascover slip 111, could possibly be used. The reflected beam re-enters theobjective lens 118, again displaced from the central optical axis.Because the range-finding beam enters the objective lens off-axis, thereflected range-finding beam emerges from the objective lens laterallydisplaced from the incident beam. The magnitude of this displacementdepends on the distance of the reflective surface, at the point ofreflection, from the objective lens. The reflected beam may also emergefrom the objective lens slightly aparallel to the incident beam, withthe difference in beam direction also depending on the distance of thereflective surface from the lens. The displacement of the reflected beamcan therefore be used to control the distance of the objective lens fromthe reflective surface, in an autofocus mechanism.

The displacement is preferably detected by a suitable optical detectorand electrical circuitry used to control the z-axis drive 119 of theobjective lens 118 accordingly. A feedback loop may be implemented bythe circuitry to ensure that the objective lens remains a constantdistance from the part of the sample in the region of interest.

Preferably, the incident range-finding beam entering the objective lens118 is substantially parallel to the optical axis of the lens.Preferably, also, the incident beam entering the lens is slightlydivergent or convergent so that the intensity of the beam at thereflective surface is reduced by being slightly out of focus. Thisreduces breakthrough of the range-finding beam, as well asphoto-bleaching of the sample.

A particular embodiment of the above discussed autofocus mechanism isillustrated in FIG. 7. For clarity, only those components common to FIG.6 which are required by the following discussion are shown in FIG. 7.

An autofocus laser 200 generates an incident range-finding laser beam202 having a width of about 1 to 2 mm, which propagates towards anuncoated pellicle beamsplitter 204. The pellicle beamsplitter 204reflects about 10% of the incident beam 202 and transmits about 90%irrespective of polarization. The transmitted light is not used.

The portion of the incident beam 202 reflected from the pelliclebeamsplitter 204 is directed to a half wave plate 206 which allows thepolarization of the beam to be adjusted. The half wave plate 206 must beselected as appropriate for the wavelength of the beam produced by thelaser 200. The incident beam 202 emerging from the half wave plate 206is directed to the objective lens 118 by a dichroic filter 208. Thedichroic filter 208 couples the incident range-finding beam 202, havinga first wavelength, into the objective lens 118, while allowingfluorescence or other image light, having a second wavelength, from thevicinity of the sample surface 116 to be transmitted to the CCD camera.In one embodiment, the objective lens is a Nikon Plan fluor, 100×magnification, NA 1.3, working distance 370 microns. This compound lenshas a rear aperture of about 6 mm and a sample side aperture of about 1mm. In such an embodiment, the incident beam 202 may have a power ofabout 20 μW and a diameter of about 1 to 2 mm as it enters the objectivelens 118.

The incident range-finding beam is directed non-centrally or off-axisinto the objective lens 118, but preferably approximately parallel tothe central axis of the lens. The beam may be only slightly off centerso that the central axis of the lens lies within the beam or close tothe center of the beam. Alternatively, the incident beam may be moresignificantly off center, for example such that the beam lies outsidethe central axis of the lens.

The incident range-finding beam 202 is directed to the reflectivesurface by the objective lens 118, and is at least partially reflectedto form a reflected beam 210 which is collected by the objective lens.The reflected range-finding beam 210 emerging from the objective isreflected by the dichroic filter 208, passes through the halfwave plate206 and is incident upon the pellicle beam splitter 204. About 90% ofthis beam is transmitted through the pellicle beam splitter 204 and isfocused using detector lens 212 onto a light sensitive element or arrayof elements 214.

In a preferred embodiment, the light sensitive element 214 is a quadcell located about 1 m from the reflective surface adjacent to thesample, and the detector lens 212 has a focal length of about 30 cm. Thequadcell is aligned such that two of its elements are spaced apart in adirection parallel to the direction of movement of the reflectedrange-finding beam 210 as the reflective surface 111 moves towards oraway from the objective lens 118. As a result, a change in the distancefrom the sample to the objective leads to a change in the relativepowers of the beam portions incident on each of these two elements. Thereflected range-finding beam is preferably slightly out of focus at thedetector so that it impinges on a part of both elements at the sametime. The remaining two elements of the quadcell are unused. Electroniccircuitry coupled to the quadcell drives an activator which adjusts theposition of the objective lens 118 along its optical axis 116 thusforming a feedback loop which seeks to keep the sample 116 at the focalpoint of the lens.

Reflections of the incident and reflected range-finding beam 202, 210from the dichroic filter 208 and from the reflective surface 111 arepolarisation dependent, governed by the Fresnel equations, so that theapparatus can be adjusted to yield a reflection beam 210 which has anoptimum or maximum intensity at the light sensitive element. Inparticular, this can be achieved by rotating the half wave plate.

In the particular embodiment discussed above, a change in the separationof the objective lens 118 and reflective surface 111 of 1 micron resultsin a movement of the focused reflected range-finding beam 210 at thelight sensitive element 214 of about 200 to 500 microns, depending onhow far the light sensitive element 214 is from the focal point of thereflected beam 210. Preferably, the light sensitive element 214 is notplaced exactly at the focal point of the reflected beam 210.

The pellicle beam splitter 204 may be replaced by a polarisationdependent optic such as a polarising beam splitter which acts such thatlight of a first polarisation is transmitted and light of a secondpolarization orthogonal to the first polarization is reflected sideways.Such a component my be used to improve the separation of the incidentand reflected beams.

When a new sample 116 and slide 114 are mounted on the prism 112 andpositioned within the microscope 100, the focal plane of the objectivelens must be matched to the plane of the sample, and the footprint ofthe illumination beam 106 onto the sample must be aligned with theregion of interest or field of view of the objective lens 118. Becausebracket 122, or other illumination director controller couples thealignment of the optical path of the illumination beam 106 to themovement of the objective lens, and hence also to the focal plane of theobjective lens, initial focussing and alignment for a new sample is muchmore straightforward than in the prior art.

FIGS. 8 a, 8 b and 8 c illustrate the footprint alignment methodimplemented by the apparatus of FIGS. 5 and 6. Each figure shows aportion of a microscope slide 114 with the vertical axis of the figuresand undulations in the lower surface greatly exaggerated, and the prism112 omitted. In FIG. 8 a the focal plane of the objective lens 118 isaligned with the part of the sample plane in the region of interest. Theoptical path of the continuous or discontinuous illumination beam 106,which is reflected from the target mirror 110, passes through the prism(not shown) and slide 114 and is incident on the part of theslide-sample boundary coincident with the region of interest definedrelative to the objective lens 118.

In FIG. 8 b the slide 114 has been translated to the right, for exampleas part of a scanning process to image an extended area of the samplelarger than the field of view. A minor undulation in the lower surfaceof the slide 114 has resulted in that part of the sample now within theregion of interest retreating away from the objective lens focal plane.Because the optical path of the illumination beam 106 is incident at anangle oblique to the normal to the focal plane, and to the samplesurface, the illumination footprint has moved to the right of the regionof interest.

In FIG. 8 c, an autofocus mechanism has moved the objective lens towardsthe slide 114 to bring the sample back into focus. Correspondingmovement of the target mirror 110 has, at the same time, moved theillumination footprint back into the region of interest. In theembodiment shown in FIG. 6 the target mirror 110 is rigidly connected tothe objective lens 118, so that, as illustrated in FIG. 8 c, therelative positions of the target mirror 110 and the objective lens 118remain unchanged.

Various alternatives and variations in the embodiment described abovewill now be discussed. Various configurations of optical componentscould be used in order to adjust the path of the illumination beam tocontrol the footprint on the sample. Instead of source and targetmirrors, a carefully directed optical fibre guide, or other suitablearrangement of lenses and/or mirrors could be used. Although, in thedescribed embodiment, the optical path of the beam between the sourceand target mirrors is substantially parallel to the focussing axis ofthe objective lens, other optical path directions could be used withsuitable compensating optics, for example with a prism having adifferent base angle.

Instead of a direct rigid coupling between the objective lens and thetarget mirror, the target mirror could be actuated by an actuatorproviding the same motion as the objective lens focussing, or z-axisactuator, with appropriate electrical coupling or common drivingcircuitry.

In an alternative embodiment, the target mirror or other illuminationbeam director element is driven according to the relative movement ofthe sample surface, for example in direct response to the output of arange finder which could also control the focus of the objective lens.Such a configuration could be particularly attractive if no suitableattachment to the objective lens is available, if vibration orengineering constraints make the use of a direct coupling between theobjective lens and the target mirror impractical, or if no useable partof the objective lens moves in fixed spatial relationship with theobjective lens focal plane.

The invention is not limited in use to TIRFM applications, or even toapplications in which the illumination beam is incident from the back ofthe sample plane with respect to the objective lens.

The invention may be applied to the oblique illumination of a samplesurface in reflection mode, or in transmission modes not requiring totalinternal reflection.

1. A method for controlling the movement of a light beam in a scanningapparatus, as a function of variation in the length of the optical pathof said beam, comprising (a) directing said light beam to an angularprism, wherein said angular prism is coupled to a sample at a baseinterface; characterised in that the base angle θ_(p) of the prismsatisfies the equations:$\frac{{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )( {{\tan \; \theta_{i}\sin \; \theta_{p}} + {\cos \; \theta_{p}}} )} = 1}{\cos \; \theta_{c}{\cos ( {\theta_{p} \pm \theta_{c}} )}}$and n_(i)sin  θ_(i) = n_(p)sin (θ_(p) − θ_(c)) wherein θ_(c) is thecoupling angle required for light incident at base interface of theprism, θ_(i) is the incident angle of the light beam on the prism, n_(i)is the refractive index of the medium at the interface where the lightbeam enters the prism and n_(p) is the refractive index of the prism;and (b) moving the prism and sample relative to the light beam, whereinthe said variation in the length of said optical path of said light beamis controlled by aligning the prism so that said light beam enters saidprism normal to the face of entry thereinto, and wherein said light beamand said prism satisfy said equations.
 2. A method according to claim 1,whereinθ_(p)=θ_(c) and θ_(i)=0.
 3. A method according to claim 1, wherein saidapparatus is a total internal reflection microscopy apparatus, andincludes means for detecting interaction of the sample at the base ofthe prism with an evanescent wave formed by total internal reflection ofthe light beam at the base of the prism.
 4. A method according to claim3, whereinθ_(c)≧sin⁻¹ (n _(s) /n _(p)) and wherein n_(s) is the refractive indexof the sample medium.
 5. (canceled)
 6. A method according to claim 5,wherein said method comprises a method of total internal reflectionmicroscopy, and further comprises the step of detecting interaction of asample at the base of the prism with an evanescent wave formed by totalinternal reflection of the light beam at the base of the prism.
 7. Amethod according to claim 28, wherein said objective lens assembly iscontained in an apparatus for imaging a sample, said apparatuscomprising: an objective lens having a focal plane and an optical axis,the lens being arranged to collect light from a region of interestdefined relative to said optical axis; an illumination directorcontrollable to direct a beam of light along an optical path so as toilluminate the sample in the region of interest at an angle oblique tosaid optical axis; and an illumination director controller arranged tocontrol the illumination director relative to the sample such that theoptical path remains directed to the sample in the region of interestfollowing replacement of the sample, or movement of the sample relativeto the objective lens.
 8. The method according to claim 7, wherein saidapparatus further comprises an angular prism having a base and a sideface having a base/side face angle θ_(p) therebetween, the sample beingadjacent to the base of the prism, the beam of light being directedthrough the side face of the prism at an angle of incidence θ_(i) so asto illuminate the sample at the base at a coupling angle θ_(c).
 9. Themethod according to claim 8 wherein the base/side face angle θ_(p)satisfies the equations:$\frac{{( {{\cos^{2}\theta_{c}} - {\sin^{2}\theta_{p}}} )( {{\tan \; \theta_{i}\sin \; \theta_{p}} + {\cos \; \theta_{p}}} )} = 1}{\cos \; \theta_{c}{\cos ( {\theta_{p} \pm \theta_{c}} )}}$and n_(i)sin  θ_(i) = n_(p)sin (θ_(p) − θ_(c)).
 10. The methodaccording to claim 9 whereinθ_(p)=θ_(c) and θ_(i)=0.
 11. The method according to claim 7 wherein theillumination director controller adjusts the illumination directorsynchronously with movements parallel to the optical axis of the samplewithin the region of interest.
 12. The method according to claim 11wherein the position of the illumination director relative to the samplewithin the region of interest remains substantially constant.
 13. Themethod according to claim 7 wherein the illumination director controlleradjusts the illumination director synchronously with movements of thefocal plane in a direction parallel to the optical axis and relative tothe sample such that the optical path remains directed to theintersection of said region of interest and said focal plane.
 14. Themethod according to claim 7 further comprising an autofocus arrangedsuch that the focal plane of the objective lens tracks the sample in theregion of interest.
 15. The method according to claim 14 wherein theautofocus projects an incident range-finding beam of light through theobjective lens such that the incident range-finding beam is incident onand reflects from the sample or a surface fixed relative to said sample,to thereby form a reflected range-finding beam collected by saidobjective lens, and detects a displacement of a point of incidence ofsaid reflected range-finding beam in a detection plane.
 16. The methodaccording to claim 15 wherein the incident range-finding beam enters theobjective lens displaced from the optical axis.
 17. The method accordingto claim 15 wherein the autofocus further comprises a detector and anelectrical autofocus coupling to drive the objective lens along theoptical axis in response to the displacement of the point of incidenceof said reflected range-finding beam in said detection plane.
 18. Themethod according to claim 7, wherein at least a part of the objectivelens is moved along the optical axis so as to bring a part of the samplewhich is within the region of interest into focus, and the illuminationdirector controller comprises a coupling that adjusts the illuminationdirector synchronously with the movement of the at least part of theobjective along the optical axis.
 19. The method according to claim 18wherein the coupling is arranged such that the position of theillumination director relative to that of the at least part of theobjective lens remains substantially constant.
 20. The method accordingto claim 7 wherein the illumination director controller comprises adirect mechanical coupling.
 21. The method according to claim 7 whereinthe illumination director controller comprises an electrical coupling.22. The method according to claim 7 wherein the objective lens ispositioned on a first side of the sample, and the illumination directoris positioned on the opposing side of the sample.
 23. The methodaccording to claim 7 wherein the apparatus is a total internalreflection fluorescence microscope.
 24. The method according to claim 23wherein the total internal reflection fluorescence microscope comprisesan optic, the sample being situated adjacent to the base of the opticand the illumination beam entering a side face of the optic so as toilluminate the sample, the base/side face angle of the optic beingsubstantially the same as the angle of internal reflection of theillumination beam at the base.
 25. The method according to claim 7wherein the illumination director is a first mirror.
 26. The methodaccording to claim 25 wherein the light beam is directed onto the firstmirror by a second mirror, the second mirror being provided with a fineadjust mechanism.
 27. A method of automatically correcting theillumination footprint of the optical path of a light beam onto amicroscopy sample surface, comprising the step of adjusting the opticalpath synchronously with changes in the position of the sample surface orthe position of an element which moves synchronously with the samplesurface.
 28. The method of claim 27 wherein the element which movessynchronously with the sample surface is at least a part of an objectivelens assembly arranged to form an image of the sample surface.
 29. Themethod of claim 27 wherein the objective lens assembly comprises anobjective lens having a focal plane and an optical axis, the lens beingarranged to collect light from a region of interest defined relative tosaid optical axis, the method further comprising directing a beam oflight along an optical path so as to illuminate the sample in the regionof interest at an angle oblique to said optical axis, wherein theoptical path remains directed to the sample in the region of interestfollowing replacement of the sample, or movement of the sample relativeto the objective lens.